凋亡抑制基因survivin表达载体的构建及其在HepG2中的表达

目的:克隆细胞凋亡抑制蛋白Survivin(SVV)的编码序列,构建含SVV基因的真核细胞表达载体并在肝癌细胞系HepG2中表达,为进一步研究该基因在肝癌发病中的作用奠定基础. 方法:根据已发表的SVV基因的核苷酸序列设计并合成一对引物,以HL-60细胞系抽提的RNA为模板进行逆转录聚合酶链反应(RT-PCR),扩增产物用BamHI和XhoI双酶酶切后定向克隆到真核细胞表达载体pcDNA3.0中,用限制性内切酶酶切重组质粒pcDNA3.0-SVV和DNA序列测定进行鉴定. 用脂质体法将pcDNA3.0-SVV导入肝癌细胞系HepG2中,G418选择培养,经免疫组化法和RT-PCR鉴定其表达. 结果:RT-PCR扩增出长445 bp的特异性片段,经克隆至pcDNA3.0后酶切鉴定证实,并测序表明序列与GenBank报道完全一致. pcDNA3.0-SVV在HepG2细胞中有稳定表达. 结论:成功克隆了SVV的编码序列,构建了其真核细胞表达载体pcDNA3.0-SVV,有助于对SVV基因在肝癌发生中的致瘤机制做进一步研究.

Construction of survivin gene vector and its expression in human hepatocellular carcinoma cell line HepG2

AIM: To clone coding sequence of survivin (SVV) gene and to construct its eukaryotic expression vector for exploring the roles of SVV gene in the carcinogenesis of hepatocellular carcinoma. METHODS: SVV gene was amplified from HL 60 by RT PCR and the fragments of the cDNA were cloned into eukaryotic expression vector pc DNA3.0 by ligating the fragments into BamHI and XhoI site. The recombinant plasmid pcDNA3.0 SVV was identified by DNA sequence and restriction analysis. The gene transfection mediated by the lipofectin was used to introduce the eukaryotic expression vector of pcDNA3.0 SVV into human hepatocellular carcinoma cell line HepG2. After selected with G418, resistant colonies were obtained. The immunohistochemical staining and RT PCR was analyzed. RESULTS: A 445 bp DNA fragment was amplified with RT PCR. Sequence and restriction analysis showed that the recombinant plasmid pcDNA3.0 SVV was constructed successfully. CONCLUSION: Human SVV gene has been successfully cloned. The sequence analysis reveals that it is 100% homologous to data published in GenBank. The construction of the recombinant plasmid pcDNA3.0 SVV will be helpful in the further study of this SVV gene in the development of hepatocellular carcinoma.